Dataset Information

Proteome and glycoproteome analysis of fibroblasts from patients with congenital disorders of glycosylation

ABSTRACT: In the biological systems, several genes are involved in protein glycosylation pathway. Congenital Disorders of Glycosylation (CDG) is known to be a multisystem disorder. Pathogenic variants in the glycosylation genes lead to abnormal N- or O-linked glycosylation that causes cellular stress. We used TMT-based N-glycoproteomics and proteomics on different patient-derived CDG and control fibroblasts to characterize the alteration in glycoproteome and proteome. 12 fractions of enriched glycopeptides after size exclusion chromatography (SEC) and 12 fractions after basic reverse phase liquid chromatography (bRPLC) were analyzed by LC-MS/MS for glycoproteomics and proteomics, respectively. A site-specific aberrant glycosylation was observed for several proteins such as mitochondrial, autophagy, extracellular matrix and cell adhesion proteins. By using quantitative proteomics, we observed alteration in abundances of several proteins separated the affected individuals and controls. The glycoproteomics and proteomics analysis in CDG patients provides the potential biomarkers and will increase our general understanding of its pathogenesis.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Fibroblast

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD044327 | Pride | 2023-10-24

REPOSITORIES: Pride

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Publications

Interplay of Impaired Cellular Bioenergetics and Autophagy in PMM2-CDG.

Ligezka Anna N AN Budhraja Rohit R Nishiyama Yurika Y Fiesel Fabienne C FC Preston Graeme G Edmondson Andrew A Ranatunga Wasantha W Van Hove Johan L K JLK Watzlawik Jens O JO Springer Wolfdieter W Pandey Akhilesh A Morava Eva E Kozicz Tamas T

Genes 20230804 8

Congenital disorders of glycosylation (CDG) and mitochondrial disorders are multisystem disorders with overlapping symptomatology. Pathogenic variants in the PMM2 gene lead to abnormal N-linked glycosylation. This disruption in glycosylation can induce endoplasmic reticulum stress, contributing to the disease pathology. Although impaired mitochondrial dysfunction has been reported in some CDG, cellular bioenergetics has never been evaluated in detail in PMM2-CDG. This prompted us to evaluate mit ...[more]

PMID: 37628636

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Project description:The most common congenital disorder of glycosylation (CDG) is caused by pathogenic variants in PMM2 (PMM2-CDG) that reduce phosphomannomutase activity and impair N-glycan synthesis. Patients present early in life with multi-system disabilities. Traditional diagnosis relies on measuring carbohydrate-deficient transferrin (CDT) and confirmation by genetic testing. However, tests for CDT can be normal in some patients, or normalize with age. Because patients also show subtle alterations in serum glycan structures, we considered that site-specific glycosylation changes might be diagnostic. We analyzed serum from 35 individuals with PMM2-CDG for discovery and validation of glycoproteomic changes. We carried out mass spectrometry-based discovery studies to profile the glycoproteome in an initial set of well-characterized affected individuals and matched controls. Next, we developed a streamlined method for analyzing additional affected individuals. Finally, targeted mass spectrometry was used to validate selected N-glycopeptides in an independent set of samples in a blinded fashion. Of the 3,342 N-glycopeptides derived from 284 glycoproteins identified in discovery experiments, individuals with PMM2-CDG exhibited a general decrease in complex-type N-glycans and an increase in truncated, mannose-rich and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man5GlcNAc2, which was not detected in controls. Notably, this glycopeptide was also detected in five affected individuals with normal CDT, including one individual after liver transplant, two individuals with a known genetic variant associated with mild disease and two individuals with well characterized variants, indicating that this glycopeptide is more sensitive than CDT. Finally, this marker was also detected by targeted analysis in two individuals with variants of uncertain significance (VUS) in PMM2 gene. Overall, we believe that C4-derived Man5GlcNAc2 glycopeptide could serve as a novel next-generation biomarker for more accurate diagnosis and tracking progress of patients in therapeutic trials.

Dataset Information

Proteome and glycoproteome analysis of fibroblasts from patients with congenital disorders of glycosylation

Publications

Interplay of Impaired Cellular Bioenergetics and Autophagy in PMM2-CDG.

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